Method for conducting platelete aggregation analysis by a cartridge device

ABSTRACT

Methods and devices for conducting platelet aggregation analysis. A method for conducting platelet aggregation analysis by a cartridge device, including providing a blood sample in the cartridge device, stirring the blood sample within the cartridge device, measuring the electrical impedance between electrodes to obtain measured electrical impedance values, comparing measured electrical impedance values, discarding and repeating the measurements of the electrical impedance in case a variation of the measurements is outside a predetermined threshold range, or reporting the measured electrical impedance values in case the variation of the measurements is within the predetermined threshold range. Reported measured electrical impedance values indicate platelet aggregation in a blood sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application which claims the benefit ofand priority to U.S. utility patent application Ser. No. 10/583,062filed Jan. 31, 2007, now U.S. Pat. No. 7,901,629, issued on Mar. 8, 2011which claims priority to PCT/EP2003/014329 filed Dec. 16, 2003, theentire contents of which are hereby incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to a cartridge device for blood analysisand a method for testing platelet functions.

BACKGROUND ART

Though the present invention can be used in many fields of measuringfluids it will be described in regard to measuring the platelet functionof blood in the following.

Blood consists of cells suspended in so called plasma, a protein richfluid. The major groups of cells in the blood are red cells, white cellsand platelets. The platelets are responsible for plugging gaps or holesin the blood vessel wall. This is achieved by a mechanism calledaggregation-adhesion reaction. When the platelets aggregate, they becomesticky and, as a result thereof, they stick to each another and to thedamaged tissue. Usually this happens when the platelets come intocontact with certain materials and chemicals, especially those relatedto damaged cells.

Platelet adhesion to injured blood vessels is an essential property inorder to close wounds and thus to ensure survival of the organism afterfor example a trauma. However the adhesion and aggregation of plateletscan also be extremely dangerous when the platelets mistake an aged orinflamed vessel for a vascular injury and thus impair blood flow intissues of vital importance. Such processes take place during amyocardial infarction or a stroke, diseases which account for moredeaths in the industrialised nations than infectious diseases or cancer.

An increasing number of patients who have suffered myocardial infarctionor stroke as well as patients who are at high risk for these events istreated for a reduced tendency of their platelets to aggregate withsubstances called “anti-platelet agents”. Besides their beneficialeffect—to reduce the incidence of platelets closing vitally importantvessels—these drugs may also induce bleeding. However a larger danger isdue to the fact that in some of the patients the drugs seem not to workproperly. Current studies have shown that up to 25% of the patientstreated do not adequately respond to this treatment.

It is thus not only of scientific interest, but also of high clinicalimportance to be able to test the function of the platelets and theindividual response to drugs which interfere with their activation.Several techniques according to the prior art are used to analyseplatelet functions or the action of anti-platelet drugs.

An early but still widely used development is the Born aggregometerwhich measures the change of light transmission of platelet-rich plasma(PRP) during the process of aggregation. Platelet rich-plasma isobtained by centrifugation of anticoagulated blood at a relatively lowspeed, which removes the heavy (hemoglobin-filled) red cells from theplasma, but leaves the much lighter platelets in the solution. Becauseof the platelet content the light transmission of PRP is relatively low.When the platelets aggregate the optical density is reduced, because theplatelets form few large aggregates, which interfere much less with thelight transmitted through the sample.

A disadvantage of this technique is the necessity of producing PRP,whose extraction is a complicated, time-consuming and thus expensiveprocedure. Furthermore, the aggregation of platelets is not measured inits natural environment, blood, thus the influence of red and whitecells on the platelets is not measured.

Other methods disclosed in documents U.S. Pat. No. 4,604,894 of Kratzerand Born, U.S. Pat. No. 6,010,911 of Baugh et al. and U.S. Pat. No.5,922,551 of Durbin et al. require relatively complex and expensivecartridges interfering with the use of these techniques for routinetesting.

Document U.S. Pat. No. 4,319,194 of Cardinal et al. discloses a plateletaggregation analysis typically performed in whole blood by measuring theelectric impedance between two electrodes, being immersed in a sample.During initial contact with the blood or PRP, the electrodes are coatedwith a monolayer of platelets. When an aggregating agent is added,platelets gradually accumulate on the monolayer coating, increasing theimpedance between the electrodes. The change in impedance is recorded asa function of time. It is preferred that the electrodes compriseprecious metals since base metals drift in blood-saline mixtures.

One disadvantage of precious metal electrodes is high costs. Hence theyare too expensive to be disposable. Therefore, the electrode assemblymust be cleaned by hand between tests, exposing the operator to contactwith the sample, and thus potentially exposing the operator to diseasestransmitted through the fluids contained in the sample. Since diseasessuch as hepatitis and AIDS can be transmitted through handling of bloodproducts, there is an understandable reluctance on the part of medicalprofessionals to handle blood, blood products and objects contaminedtherewith.

A further disadvantage of this structure is due to the fact that theelectrodes of the aggregometer have to be handled by the user during thecleaning procedure, potentially disturbing the adjustment of thedistance between the electrodes, causing inconsistent results.Furthermore each electrode requires exact placement of the wires duringfabrication, making the final product expensive.

Document U.S. Pat. No. 4,591,793 of Freilich describes a substitution ofthe wire electrodes by a conductive ink printed on a plasticnon-reactive base.

However this device is detrimental due to the fact that the plateletshave difficulties in adhering to the exposed conductive surface of theFreilich device. Sometimes the aggregated platelets break off thesurface, causing a sudden change in impedance. Hence the measuredresults by the device are inconsistent and not reproducible.

A further measuring cell assembly according to the prior art isdisclosed in Document U.S. Pat. No. 6,004,818 of Freilich et al., whichis shown in FIGS. 11A and 11B.

FIG. 11A illustrates an explosive view of a part of a measuring celldevice comprising an insulator 1, which is sandwiched between twoflag-shaped electrodes 2. Each electrode 2 includes a connection tab 3at one end and a tip 4 at the other end thereof, with a shaft 5 joiningthe tab 3 and the tip 4 respectively. After joining the two electrodes 2and insulator 1 together a non-conductive coating is applied to theinsulator 1 and to the electrode shafts 5.

FIG. 11B illustrates a perspective view of a measuring cell deviceaccording to the prior art. As shown in FIG. 11B the electrode assemblyis fixed within a cuvette 56 using a positional clip 7. Prior to andduring measurement, a stir bar 8 is activated to generate a circularflow of sample within the cuvette 6.

One drawback of the aforementioned measuring cell device is the use ofpunched sheet metal for the electrodes 2. The outline of the electrodescan economically be produced by the process of punching or relatedmethods. However the surface qualities produced by these methods arerelatively poor and vary during the production of large quantities(because of the aging of the punching blades applied during theprocess). The quality of the surfaces, which strongly affect themeasurement, are thus strongly varying resulting in high variationsbetween different disposable electrodes.

A further disadvantage is due to the fact that the complete measuringcell device consists of six different pieces, namely the two electrodes3, the insulator 1, the coating, a cuvette 6, a positional clip 7 and astir bar 8. This results in an expensive, complicated productionprocess, which is either labor intensive or requires high investmentsfor an automated assembly line.

Additionally the measuring cell device of Freilich does not overcome therelatively high variation reported for whole blood aggregometry. Indocument U.S. Pat. No. 6,004,818 a variation of around 10-15% betweenmultiple measurements is reported for said measuring cell assembly.

SUMMARY OF THE INVENTION

Hence it is an object of the present invention to provide a cartridgedevice which overcomes the above mentioned drawbacks, particularly toprovide a cartridge device which is economical, reproducible, safe andeasy to use.

It is another object of the present invention to eliminate the limitedreproducibility of previous methods according to the prior art for themeasurement of aggregation in whole blood.

It is still another object of the present invention to provide acartridge device which is accurate and so economical to produce that itcan be discarded after each test.

It is still another object of the present invention to provide acartridge device which can be provided ready to use, so the applicationof the technique is simple and contains only few user-related sources oferror and variation.

The present invention provides a cartridge device for analysing bloodcomprising a cell having a receiving portion for receiving a bloodsample and a jack portion for receiving a plug; a stirring device forcirculating said blood sample within said receiving portion; and anelectrode holder having at least one incorporated electrode wire pair;wherein the electrode holder is attachable to the cell such that one endof the at least one electrode wire pair forms a sensor unit formeasuring the electrical impedance between the two electrode wires ofthe at least one electrode wire pair within the blood sample and thatthe opposite end of the at least one electrode wire pair forms a plugportion being connectable directly to the plug for an electricalconnection of the sensor unit to an analyser.

Furthermore the present invention provides a cartridge device foranalysing blood comprising a cell having a receiving portion forreceiving a blood sample; a stirring device for circulating said bloodsample within said receiving portion; and at least two electrodes formeasuring the electrical impedance between the at least two electrodeswithin the blood sample; wherein the at least two electrodes consist ofa metal comprising a first material with a high electrical conductivity,which is covered by a second material, which has a high electricalconductivity and which is resistant against oxidation.

Additionally the present invention provides a method for analysing bloodby means of a cartridge device comprising at least three electrode wiresor electrodes for measuring the electrical impedance between at leasttwo of the at least three electrode wires or electrodes, comprising thefollowing steps: measuring the electrical impedance between at least twodifferent pairs of electrode wires or electrodes; comparing the measuredelectrical impedance values; discarding and repeating the measurementsin case the variation is outside a predetermined threshold range; orindicating the measured electrical impedance values and/or the meanvalue thereof in case the variation is within the predeterminedthreshold range.

The dependent claims disclose further features and advantages of thepresent invention.

Hence the present invention provides a disposable device which overcomesthe main problems of the aforementioned developments. The apparatusmeasures the change of impedance during the process of aggregation,whereas the quality of the measurement is ensured by a double-, triple-or multi-determination of the impedance. The cartridge can bemanufactured at very low costs and is well suited for full automation ofthe production process.

The device for monitoring blood-platelet aggregation comprises acartridge including electrodes; a means for stirring the sample; a powersource for supplying electric current to the electrodes; and a dataanalysis device for receiving and analysing the change in electricalresistance or impedance between the electrodes.

A device is provided which is economical, accurate andquality-controlled. The test includes a disposable test cell havingpreferably at least two separate sensor elements. Each sensor elementconsists of preferably at least two separate conductive elements. Duringthe analysis blood is placed in the test cell. By adequate means it isstirred. Optionally reagents activating or inhibiting platelet functionare added. When blood platelets are activated they adhere on theconductive elements and alter electrical variables measured e.g. theresistance between the conductive elements or the electrical impedance.By comparing the values assessed on the different electrodes the resultof the analysis is being controlled. When an adequate accordance betweenthe individual results is attained, the result is accepted and the valueor the mean (or median) value of the results is reported. If highlydiscrepant results are determined the analysis is rejected and arepeated analysis is requested.

The present invention further describes an economical and standardizedproduction technique using injection moulding providing an improvedanalytical signal using suitable electrode compositions. The conductiveelements are formed by wires which incorporate two functions. In theirupper part they form a connector—or jack—by which the conductivitysignal is detected. In the lower part they are contacted with the bloodsample and serve as a sensor for the platelet aggregation. Theconductive elements are formed from a material of high conductivity andprecious surface. A silver-copper material coated by silver or otherprecious material is preferably applied. This leads to a strong signal,due to the higher difference in conductivity between the platelet-coatedvs. the uncoated electrode. In addition also the detection of theconductivity over the connector—or jack—is improved.

Hence the present invention allows to asses the whole blood aggregationin a routine setting. Unlike previously applied methods for theassessment of whole blood aggregation it is easy to use and does notrequire the cleaning of blood-contaminated devices. Compared to themeasurement of aggregation in platelet rich plasma the inventive methodprovides the advantage to eliminate the need to centrifuge blood toobtain PRP for use in measuring aggregation of platelets optically. Thusreduced labor costs, increased speed, and test of the platelets in theirnatural milieu are important advantages. The measurement in whole bloodalso allows studies to be performed in cases where optical aggregationis not reliable, such as giant platelets (Bernard-Soulier syndrome),where red cells have been lysed or where it is difficult to obtainenough blood to make PRP, such as with small animals or babies.

In addition whole blood aggregometry has the advantage that—like theBorn aggregometry—it provides a continuous signal of the kinetics ofplatelet aggregation, unlike other methods, which provide only a measurefor total aggregation, without revealing the kinetics of the process.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be further understood by reference to the drawings,wherein:

FIG. 1 is a perspective view of a cell according to an embodiment of thepresent invention;

FIG. 2 is a perspective view of an electrode holder according to anembodiment of the present invention (with double determinationtechnique);

FIG. 3 is a side view of an electrode holder according to certainembodiments of the present invention (double, linear triple or linearquadruple detection technique);

FIG. 4 is a side view of the cartridge according to certain embodimentsof the present invention (double, linear triple or linear quadrupledetection technique);

FIG. 5 is a top plan view of a cartridge according to a first embodimentof the present invention (with double determination technique);

FIG. 6 is a perspective view of the cartridge according to the firstembodiment of the present invention (with double determinationtechnique);

FIG. 7 is a sectional view of a cartridge device with attached plug andcable according to an embodiment of the present invention;

FIG. 8 is a perspective view of a cartridge device according to a secondembodiment of the present invention (with triple determinationtechnique;

FIG. 9 is a perspective view of a cartridge device according to a thirdembodiment of the present invention (with linear quadruple determinationtechnique);

FIG. 10 is a top plan view of a cartridge device according to a fourthembodiment of the present invention (with circular triple determinationtechnique;

FIG. 11A is an explosive view of a part of a cartridge device accordingto the prior art; and

FIG. 11B is an perspective view of a cartridge device according to theprior art.

DETAILED DESCRIPTION OF THE INVENTION

In all figures like reference numerals are used to demote like elementsand/or like functions of respective elements as long as nothing else isstated. It should be noticed that in the following the term cartridgedescribes the disposable structure applied in preferred embodiments ofthe invention. It consists of a cell 9 and an electrode holder 14.

In a preferred embodiment the cartridge device 20 consists of two mainparts 9 and 14 made by particularly injection moulding. The one part isa single pieced receiving means and subsequently called cell 9.

FIG. 1 depicts a cell 9 in a perspective view according to a preferredembodiment of the present invention. The cell 9 comprises at least apreferably cylindrical receiving portion or cup portion 10, which isopen at one face side, holding a sample during the analysis. The sampleis placed into the cup portion 10 of the cell 9 by means of aparticularly conical funnel tube 11.

The cell 9 further comprises a jack portion 12 adjacent to the cupportion 10 and separated therefrom by a stopping wall 27. Together withan electrode holder 14, described later in further details (see FIG. 2),the jack portion 12 forms a jack 18 (see FIG. 3), in which a plug 22(described later in further details) is arranged and which allows theelectrical connection of the cell 9 to an instrument, for example ananalyser.

A guiding rail 13 is formed on both sides of the funnel tube 11 of thecell 9, respectively, for guiding the electrode holder 14 into a secureand tight connection with the cell 9 as shown in FIG. 4.

Preferably and as shown in FIG. 1 the whole cell 9 is formed in a waythat it can be easily unmoulded with a two part injection mouldingmould. This requires that the cup 9 does not form any undercuts and thatthe outer and inner surfaces are at least slightly conical. Although thecell 9 has three different functional portions, it can inexpensively beproduced as one part using injection moulding. This minimises productioncosts on the one hand and the need for manual handling for the use onthe other hand.

The cell 9 is in particular produced of blood compatible material suchas polystyrene. Other usable materials are polymethyl methacrylate(PMMA) or polyethylene. The importance of using a blood compatiblematerial is that the blood platelets will not get activated by thecontact with the cell material. This allows to activate specifically theplatelets as intended for the different test methods performed.

Dual Electrode Cartridge Device

The other part of the cartridge device 20 is the electrode holder 14,illustrated in FIG. 2 in a perspective view according to a firstembodiment of the present invention. The electrode holder 14 consistsparticularly of a plastic body 15, in which two tiny electrode wirepairs 16, 24 are incorporated. Each wire of a wire pair 16, 24 haspreferably a diameter of about 0.1-0.5 mm, most preferably 0.3 mm.

The electrode holder 14 comprises especially a L-formed body 15 with along part 15 a and a short part 15 b perpendicular to the long part 15a. At the face side of the long part 15 a of the electrode holder 14each electrode wire of the electrode pairs 16, 24 protrudes and forms asensor portion 16 a, 24 a. For example, two sensor portions 16 a or 24 aform together a sensor unit 17 a or 17 b. During the analysis the sensorunits 17 a and 17 b are completely immersed into the blood sample. Theblood platelets adhere on the sensor portions 16 a, 24 a of the wirepairs 16, 24 and change the electrical impedance between said two wiresof a respective wire pair 16 or 24. These measured impedance values canbe compared with each other and/or with a predetermined threshold.

At the face side of the short part 15 b of the electrode holder 14 eachelectrode wire of the electrode pairs 16, 24 protrudes under apredetermined angle, for example 50°, and forms a connector portion 16b, 24 b, respectively.

The electrode holder 14 of this preferred embodiment comprises twolinear independent sensor units 17 a, 17 b, each formed by an electrodewire pair 16, 24. The sensor units 17 a, 17 b are particularly placedsymmetrically to each other at the face side of the long part 15 a ofthe body 15 in order to ensure identical flow conditions around eachwire pair and acceptable measurement results. Preferably the two wiresof an electrode pair 16 (24) are positioned parallel to each other andparallel to the wires of another electrode pair 24 (16).

The electrode holder is preferably inexpensively produced by injectionmoulding. This requires to insert the wires of the electrode pairs 16,24 into the mould and to extrusion-coat them by the resin. This posesthe problem not to bend the thin wires by the high pressure in theinjection mould. Also it is important to automate the process of placingthe wires into the mould, thus allowing fully automated production andthus low costs. Preferably the wires are automatically placed into themould from a roll, then extrusion coated and afterwards automaticallytaken out of the mould and then cut into the right dimensions. This alsoprovides the advantage that cable on a roll is less expensive thanpre-cut cable bars. It is advantageous to keep the wires under tensionduring the injection process in order to prevent bending of the thinwires.

It is also advantageous to keep the body 15 of the electrode holder 14thin, as it is shown in FIG. 2. This leads to reduced pressurerequirements in the injection process and thus also to a lower tendencyto bend the wires of the electrode wire pairs 16, 24. A thickness of 1-5mm of the body 15 is preferred most. The plastic material used for theelectrode holder is preferably a blood compatible material such aspolystyrene, PMMA or polyethylene, most preferably polystyrene.

The material selected for the electrodes or wires needs to fulfilseveral requirements. It should provide a low electrical resistance.This provides a good electrical connection of the wires in theconnection portions 16 b, 24 b to plugs 22 or equivalent devices and inaddition it provides a stronger signal of the analysis. In the sensorportions 16 a, 24 a the wires are coated by the activated plateletswhich enhances the electrical resistance between them. When wires of alower resistance are applied this leads to a stronger resistance changewhen the wires are coated by the platelets compared to a material withlower conductivity. Materials with a high conductivity include forexample copper and copper alloys (copper-silver alloy, copper-magnesiumalloy).

However the wire material must also not oxidate when contacted to blood,even when different oxidating drugs are present. This requires thesurface of the wires 15 to have a low tendency to oxidate. Suchmaterials are for example precious metals such as platinum, gold,silver. The named materials with a high conductivity (copper and copperalloys) have a high tendency to oxidate. However the precious metals aretoo expensive to be used for the production of a single use cartridge.According to the present invention it was found that by coating a lowcost wire material with a high conductivity (preferably a silver-copperalloy comprising 0.2-2% silver, most preferably 0.9% silver) with aprecious material such as silver (preferably using a coating of 0.5-20g/kg, most preferably 2 g/kg) a wire is obtained which is economical,which provides a good electrical impedance and which is alsosufficiently resistant to oxidation during the analysis. Other coatingsmade of gold, platinum or other precious metals can be also applied.

The electrodes are preferably wires having a circular cross-section. Theinventors have tested diameters between 0.1 mm and 0.50 mm. The signalturns out to become weaker with increasing diameter, so that theelectrodes should be thin. The electrode holder is manufactured byinjection moulding which requires tearproof rods, i.e. the rods shouldnot be to thin. The optimal value for diameter of the electrodes whichencompasses these two aspects turned out to be 0.3 mm.

The length of the ends of the electrodes which stand out of theelectrode holder 14 is preferably 4 mm. The inventors have testedlengths between 2 mm and 6 mm. It turned out, that the longer the endsare, the weaker the signal becomes, so that short ends are preferred.However, when the ends become too short, the production of the electrodeholder becomes more complicated, so that an optimal length is about 4mm.

A spacing of 0.5-1 mm between the electrode wires was found to provideoptimal signal and reproducibility.

The electrode wires are preferably bended two times as is best seen inFIGS. 3 and 4. This bending ensures that the ends of the electrodes formtogether with the jack portion 12 of the cell 9 a jack. In the presentcase the inventors use preferably a geometry consistent with the normedjack RJ12. This allows to use the standard RJ12 plug to connect thecartridge device 20 to an analyser just by putting the plug into thejack portion 18.

After the injection moulding process the wires 16, 24 are bended forexample under an angle of at least 90° as shown in FIG. 3.

Afterwards the electrode holder 14 is connected to the cell 9 by guidinga guiding part 28 of the holder 14 in said respective guiding rails 13of the funnel tube 11 of the cell 9 until it contacts the upper edge ofthe stopping wall 27.

FIGS. 4 to 6 illustrate an assembled cartridge device 20 in a sectionalside view, a top plan view and a perspective view according to apreferred first embodiment of the present invention.

A magnetic or paramagnetic stir bar 19 for stirring the blood sample isplaced into the cup portion 10. Then the whole device is packed intoappropriate means for storage and shipping to the customer.

Before the analysis the user takes the cartridge device 20 out of thepackaging and places it into an appropriate receptacle of the analyser.The receptacle is preferably heated to 37° to ensure that the analysistakes place under standardized and physiological temperature conditions.

The filling amount of the blood sample is preferably large enough toensure that the ends of the electrode wires projecting from the faceside of the long part 15 a of the body 15 are completely covered.

Appropriate means are placed under the receptacle that induce a stirringmotion of the stir bar 19. The preferred means for stirring the sampleis the use of electromagnets that are alternately turned on and off andtherefore induce a rotation of the stir bar 19. The stir bar 19 cancomprise a polytetrafluoroethylene (e.g., Teflon) coated stir bar, steelor siliconized steel. Siliconized stainless steel is the preferredmaterial, as it is less expensive than polytetrafluoroethylene (e.g.,Teflon) coated stir bars. Non-coated stir bars can alter the plateletactivation due to the contact and adhesion of platelets to thethrombogenic steel material. Also permanent magnets that are rotated byadequate means or other means for inducing rotation of the sample (suchas ultrasound, orbital movements of the cup) can be applied as obviousto a person skilled in the art.

The user then connects a plug 22, preferably a standard RJ12 plug, tothe jack 18 as illustrated in FIG. 7, which shows a sectional view of anassembled cartridge device with a connected plug 22 according to apreferred embodiment of the present invention. The plug 22 is preferablya standard plug, which comprises particularly a conductive element 22contacting the contact regions 16 b, 24 b, i.e. the plug portions 21 aand 21 b of the electrode wire pairs 16 and 24. The plug 22 is connectedto an analyser (not shown) by means of a connection cable 23 and allowsthe signal of the analysis to be transferred from the cartridge device20 to the analyser.

During the analysis the analyser continuously records the impedancechange on both sensor units 17 a and 17 b. Under optimal conditions theimpedance change of both sensor units 17 a and 17 b will be identical ornearly identical. However when due to material variations, dirt, damageof the wires 16, 24 during transport or other errors one sensor unit isdefect, a strong variation is determined between the two sensor units 17a and 17 b and the measurement is discarded. An automatic algorithm inthe software distinguishes acceptable from non-acceptable variationbetween the results of the sensor units 17 a and 17 b and automaticallystops the analysis if required. When an acceptable variation isrecorded, for example the mean value of the two determinations isreported to the user. This leads to significantly improved precision.The rate of wrong diagnosis is significantly decreased compared tosingle analysis units.

After the analysis the user disconnects the plug 22 from the cartridgedevice 20 and discards it.

Linear Triple Cartridge Device

A second preferred embodiment of the cartridge device 20 is shown inFIG. 8, which is a perspective view. The cell 9 according to the secondembodiment is identical to the previous embodiment and therefore it isreferred to the above evaluations.

However contrary to the first embodiment the electrode holder 14according to the second embodiment comprises three sensor units 17 a, 17b, 17 c, which are linearly arranged to each other. Each sensor unitconsists of a pair of electrode wires 16, 24 and 25, respectively. Dueto the linear arrangement the electrode wire pair 24 is in the middle ofthe two remaining electrode wire pairs 16 and 25 and is exposed to asignificantly lower blood flow when compared to the outer electrode wirepairs 16 and 25. Hence according to the second embodiment it is possibleto compare platelet adhesion and aggregation under varying flowconditions. It is obvious to a person skilled in the art that byvariations of the number and geometric arrangement of the test units theflow conditions under which the platelets are analysed can be varied andalso differentiated software algorithms can be applied.

Linear Quadruple Cartridge Device

According to a third preferred embodiment as shown in a perspective viewin FIG. 9, the cell 9 is identical to the previous embodiments andtherefore it is referred to the above evaluations.

However the electrode holder 14 according to the third embodimentcomprises four sensor units 17 a, 17 b, 17 c, 17 d. These sensor unitsare linearly arranged to each other. Each sensor unit 17 a, 17 b, 17 cand 17 d consists of a pair of electrode wires 16, 24, 25 and 26,respectively. Due to the linear arrangement the middle two electrodewire pairs 24 and 25 are exposed to a lower blood flow compared to theouter electrode wires 16 and 26. Hence according to the third embodimentit is possible to make a double-determination of the plateletaggregation under low and high blood flow conditions, referring to theflow conditions of blood in vessels with small and large diameter.

Circular Triple Cartridge Device

FIG. 10 illustrates a top plan sectional view of a cartridge device 20according to a fourth embodiment of the present invention. The cell 9 isidentical to the aforementioned embodiments and therefore it is referredto the above evaluations.

However the electrode holder 14 is formed to allow a circulararrangement (FIG. 8) of several sensor units. According to the presentarrangement shown in FIG. 10 three sensor units 17 a, 17 b and 17 c arearranged at the same radial location in the blood sample, i.e. in thereceiving portion 10, and exposed to the identical or nearly identicalblood flow, which allows to directly compare the signals.

According to the fourth embodiment the analyser independently recordsthe changes in conductivity between the wire pairs of the three sensorunits 17 a, 17 b and 17 c. Thus three independent results are obtained.For example the mean or median value is reported to the user, resultingin an enhanced precision of the analysis when compared to the prior artand to inventive embodiments containing only two independent sensorunits.

Surface-Coated Punched Electrodes

According to another preferred embodiment a cartridge device isprovided, wherein the electrodes are formed by punched metal (as shownin FIG. 11A), especially by using a highly electrically conductivematerial such as copper or copper alloys. The surface is coated using aprecious material with a high conductivity, such as silver, gold orplatinum. This allows to correct the aforementioned poor surface qualityof the punched material by the coating with the precious material. Inaddition the high conductivity of the used metal material (e.g. copper)improves the signal of the analysis by the higher conductivity reductionwhen the electrode surface is coated by the activated platelets.

Multiple Punched Electrodes

According to another preferred embodiment a cartridge device isprovided, wherein at least three electrodes made of punched metal are incontact with the blood. The shape of the electrodes can be similar tothe shape of the electrodes shown in FIG. 11A. At least two separatemeasurements of conductivity are parallel performed. In case threeelectrodes are used, the conductivity between electrode 1 and electrode2 is measured as signal 1. The conductivity between electrode 2 and 3 isused as signal 2. And the conductivity between electrode 3 and electrode1 is used as signal 3. In case the variation of a determinedconductivity signal is higher than an acceptable threshold (preferably20%) of the mean value of the three signals, this signal is rejected andonly the two other signals are analysed. In case also the conductivityof the remaining two signals shows a variation being too high, the wholeanalysis is discarded and the user is instructed to repeat the analysis.In case the signal variation is acceptable, the mean value or medianvalue of the signals is reported to the user. This allows asignificantly reduced probability of wrong determinations and also areduced variation of the results.

Hence the present invention provides a cartridge device which solvesseveral problems. On the one hand the limited reproducibility ofprevious methods according to the prior art for the measurement ofaggregation in whole blood is eliminated. On the other hand a cartridgeis provided which is accurate and so economical to produce that it canbe discarded after each test.

Another advantage of the present invention is due to the fact that acartridge which can be manufactured ready to use is provided, so thatthe application of the technique is simple and contains only fewuser-related sources of error and variation.

While the forgoing description and examples set forth specificillustrations of the practice of the present invention, thoseillustrations are intended to be representative only. Thus it will beapparent that numerous modifications and variations upon the designs andprocesses particularly described herein may be resorted to by thoseskilled in the art within the scope of the appended claims.

For example instead of the electrode wire pairs of the first to thirdembodiments several single electrodes or electrode wires can be used toreceive several independent and separate measurement results.

It is obvious to a person skilled in the art that the wire sections 16b, 24 b, 25 b and 26 b can serve as a plug or a jack in order to allow aconnection to an analyser.

REFERENCE NUMERAL LIST

-   1 insulator-   2 electrode-   3 connection tap-   4 tip-   5 shaft-   6 cuvette-   7 clip-   8 stir bar-   9 cell-   10 receiving portion/cup portion-   11 tunnel tube-   12 jack portion-   13 guiding rail-   14 electrode holder-   15 plastic body-   15 a long part of body-   15 b short part of body-   16 first electrode wire pair-   16 a sensor portion-   16 b connection portion-   17 a first sensor unit-   17 b second sensor unit-   17 c third sensor unit-   17 d fourth sensor unit-   18 jack-   19 stir bar-   20 cartridge device-   21 a plug portion-   21 b plug portion-   21 c plug portion-   21 d plug portion-   22 plug-   23 connection cable-   24 second electrode wire pair-   24 a sensor portion-   24 b connection portion-   25 third electrode wire pair-   25 a sensor portion-   25 b connection portion-   26 fourth electrode wire pair-   26 a sensor portion-   26 b connection portion-   27 stopping wall-   28 guiding part

The invention claimed is:
 1. A method for conducting platelet aggregation analysis by a cartridge device comprising two, three, or four pairs of electrode wires for measuring the electrical impedance between two respective electrodes of each pair of electrode wires, the method comprising the following steps: (a) providing a blood sample in a cup portion of the cartridge device; (b) stirring said blood sample for circulating said blood sample within said cup portion of the cartridge device; (c) providing an electrode holder having incorporated the two, three, or four pairs of electrode wires, wherein sensor portions of the two, three, or four pairs of electrode wires are completely immersed into the blood sample, wherein the two respective electrodes of the two, three, or four pairs of electrode wires are positioned parallel to each other and parallel to the respective electrodes of the other pairs of electrode wires; (d) measuring the electrical impedance between the respective electrodes of the at least two, three, or four pairs of electrode wires to obtain measured electrical impedance values; (e) comparing the measured electrical impedance values from the two, three, or four pairs of electrode wires immersed in the blood sample to one another; (f) discarding and repeating the measurements of the electrical impedance in case a variation of the measurements is outside a predetermined threshold range; or (g) reporting the measured electrical impedance values from the two, three, or four pairs of electrode wires and/or the mean or median value thereof in case the variation of the measurements is within the predetermined threshold range, wherein the reported measured electrical impedance values indicate platelet aggregation in the blood sample.
 2. The method according to claim 1, wherein only those measurement values are rejected, which are outside the predetermined threshold range, wherein the remaining measurement values and/or the mean values thereof are reported.
 3. The method of claim 1, wherein at least two separate measurements of impedance are performed in parallel.
 4. The method of claim 3, wherein if three pairs of electrode wires are used, the impedance between a first pair of electrode wires and a second pair of electrode wires is measured as a first impedance signal, the impedance between the second pair of electrode wires and a third pair of electrode wires is measured as a second impedance signal, and the impedance between the third pair of electrode wires and the first pair of electrode wires is measured as a third impedance signal.
 5. The method of claim 4, wherein if a variation of a determined impedance signal is higher than a predetermined threshold range of a mean value of the first, second and third impedance signals the signal is rejected, and only the remaining two impedance signals are analysed.
 6. The method of claim 5, wherein if the impedance of the remaining two impedance signals shows a variation being higher than the predetermined threshold range, the whole analysis is discarded.
 7. The method of claim 5, wherein if a variation of a determined impedance signal is lower than the predetermined threshold range of a mean value of the first, second and third impedance signals a mean value of the measured electrical impedance values is reported to a user.
 8. The method of claim 1, further comprising adding reagents that either activate or inhibit platelet function to the cup portion of the cartridge device.
 9. The method of claim 1, further comprising continuously recording an impedance change on each electrode pair of said two, three or four pairs of electrode wires.
 10. The method of claim 9, wherein if an impedance variation is detected between the two electrodes of said two, three or four pairs of electrode wires, said measurement is discarded.
 11. The method of claim 10, wherein an automatic algorithm in a software of an analyser connected to the cartridge device distinguishes between an acceptable and a non-acceptable impedance variation and automatically stops the analysis.
 12. The method of claim 1, wherein the method provides a continuous signal regarding the kinetics of platelet aggregation.
 13. The method of claim 1, wherein spacing between said wires comprised by the pairs of electrode wires is of about 0.5 to 1 mm.
 14. The method of claim 1, wherein the wires comprised by said two, three or four pairs of electrode wires consist of a metal having a low tendency to oxidize.
 15. The method of claim 14, wherein said metal comprises a first material comprising copper and copper alloy, which is covered by a second material comprising at least one of silver, platinum, and gold.
 16. The method of claim 15, wherein the first material is a copper-silver alloy.
 17. The method of claim 16, wherein the copper-silver alloy comprises 0.2% to 2% silver.
 18. The method of claim 16, wherein the copper-silver alloy comprises 0.9% silver.
 19. The method of claim 15, wherein the second material is a silver coating with a mass coverage in the range of 0.5 to 20 g/kg.
 20. The method of claim 15, wherein the second material is a silver coating with a mass coverage of about 2 g/kg.
 21. The method of claim 1, wherein said cartridge device comprises three electrode wires for measuring the electrical impedance between at least two of the at least three electrode wires. 